JOURNAL OF VIROLOGY, Nov. 2010, p. 11200–11209
Copyright © 2010, American Society for Microbiology. All Rights Reserved.
Vol. 84, No. 21
Mutation at a Single Position in the V2 Domain of the HIV-1
Envelope Protein Confers Neutralization Sensitivity to a
Highly Neutralization-Resistant Virus?†
Sara M. O’Rourke,1Becky Schweighardt,2Pham Phung,2Dora P. A. J. Fonseca,1Karianne Terry,1
Terri Wrin,2Faruk Sinangil,3and Phillip W. Berman1*
Department of Biomolecular Engineering, University of California, Santa Cruz, California 950641; Monogram Biosciences,
South San Francisco, California 940802; and Global Solutions for Infectious Diseases, South San Francisco, California 940803
Received 14 April 2010/Accepted 2 August 2010
Understanding the determinants of neutralization sensitivity and resistance is important for the develop-
ment of an effective human immunodeficiency virus type 1 (HIV-1) vaccine. In these studies, we have made use
of the swarm of closely related envelope protein variants (quasispecies) from an extremely neutralization-
resistant clinical isolate in order to identify mutations that conferred neutralization sensitivity to antibodies
in sera from HIV-1-infected individuals. Here, we describe a virus with a rare mutation at position 179 in the
V2 domain of gp120, where replacement of aspartic acid (D) by asparagine (N) converts a virus that is highly
resistant to neutralization by multiple polyclonal and monoclonal antibodies, as well as antiviral entry
inhibitors, to one that is sensitive to neutralization. Although the V2 domain sequence is highly variable, D at
position 179 is highly conserved in HIV-1 and simian immunodeficiency virus (SIV) and is located within the
LDI/V recognition motif of the recently described ?4?7 receptor binding site. Our results suggest that the
D179N mutation induces a conformational change that exposes epitopes in both the gp120 and the gp41
portions of the envelope protein, such as the CD4 binding site and the MPER, that are normally concealed by
conformational masking. Our results suggest that D179 plays a central role in maintaining the conformation
and infectivity of HIV-1 as well as mediating binding to ?4?7.
A major goal in human immunodeficiency virus type 1
(HIV-1) vaccine research is the identification of immunogens
able to elicit protective immunity from HIV-1 infection. Re-
sults from the recent RV144 clinical trial in Thailand (53) have
provided evidence that immunization with vaccines containing
the recombinant HIV-1 envelope glycoprotein gp120 (6, 7) can
protect humans from HIV infection when incorporated in a
prime/boost immunization regimen. Although the level of pro-
tection observed in the RV144 trial (31%) was modest, it
represents a significant advance in HIV-1 vaccine research and
has rekindled the efforts to identify improved subunit vaccine
antigens that might achieve even higher levels of protection. In
these studies, we have sought to understand the molecular
determinants of neutralization sensitivity and resistance in
HIV-1 envelope proteins for the purpose of developing im-
proved vaccine antigens.
In previous studies (47), we have described a novel method
of mutational analysis of the HIV-1 envelope protein, termed
swarm analysis, for identification of mutations that confer sen-
sitivity and/or resistance to broadly neutralizing antibodies
(bNAbs). This method makes use of the natural amino acid
sequence virus variation that occurs in each HIV-infected in-
dividual to establish panels of closely related envelope proteins
that differ from each other by a limited number of amino acid
substitutions. We have previously used this method to identify
a novel amino acid substitution in gp41 that conferred sensi-
tivity to neutralization by monoclonal and polyclonal antibod-
ies as well as virus entry inhibitors. In this paper, we describe
a mutation in the V2 domain of gp120 that similarly induces a
neutralization-sensitive phenotype in an otherwise neutraliza-
tion-resistant envelope sequence.
Previous studies (10, 14, 33, 40, 43, 52, 72, 74) have sug-
gested that sequences in the V2 domain act as the “global
regulator of neutralization sensitivity” and confer neutraliza-
tion resistance by restricting access to epitopes located in the
V3 domain, the CD4 binding site, and chemokine receptor
binding sites through “conformational masking” of neutraliz-
ing epitopes. Deletion of the V2 domain markedly increases
neutralization sensitivity (10, 57, 62, 74), and several envelope
proteins with V2 domain deletions have been developed as
candidate HIV-1 vaccines (5, 42, 61). In this paper, we show
that a single substitution of asparagine (N) for aspartic acid
(D) at position 179 in the C-terminal portion of the V2 domain
(corresponding to position 180 in HXB2 numbering) converts
a highly neutralization-resistant virus to a neutralization-sen-
sitive virus with a phenotype similar to that described for V2
domain deletion mutants. Position 179 has recently attracted
attention as a critical element of the ?4?7 integrin binding site
that affects virus tropism to the gut (2). Our results suggest that
mutation at position 179 results in a conformational change
that increases neutralization sensitivity by exposure of epitopes
in both gp120 and gp41 that are normally masked in the tri-
* Corresponding author. Mailing address: Department of Biomolec-
ular Engineering, Baskin School of Engineering, University of Califor-
nia, Santa Cruz, 1156 High Street, MS-SOE2, Santa Cruz, CA 95064.
Phone: (831) 459-3529. Fax: (831) 459-1970. E-mail: email@example.com
† Supplemental material for this article may be found at http://jvi
?Published ahead of print on 11 August 2010.
meric structure of gp160 and thus are unavailable for antibody
MATERIALS AND METHODS
Envelope genes and swarm analysis. Libraries of full-length envelope genes
were isolated by reverse transcription-PCR (RT-PCR) from cryopreserved
plasma samples from patients who became infected with HIV-1 while partici-
pating in the VAX004 phase 3 trial of the AIDSVAX B/B vaccine (20). The
specimens selected for analysis represented recent infections with a mean esti-
mated time after infection of 109 ? 58 days (48). A panel of clade B reference
isolates was obtained from the NIH AIDS Reagent Repository and included
JRCSF, YU2, QHO69.42, and TRO-11 (GenBank accession numbers U63632,
M93258.1, AY835439, and AY835445). The JRCSF and YU2 envelope genes
were isolated from proviral clones by PCR and cloned into an expression vector.
The QH0692.42 and TRO-11 envelopes were obtained as full-length Env/Rev
cassettes and were subcloned directly into the standard Monogram Biosciences
expression vector for pseudovirus production. The swarm analysis protocol was
described previously and is an application of the clonal analysis procedure de-
veloped by Monogram Biosciences (South San Francisco, CA) (19, 21, 22, 24,
28–32, 47). Briefly, the population of viral envelope genes present in the patient
plasma was amplified by RT-PCR and then cloned into expression vectors. To
test individual clones derived from the envelope population, the DNA was
diluted and retransformed in bacteria, and individual clones were selected and
screened for infectivity using the Monogram Biosciences coreceptor tropism
assay. Pseudotype viruses containing cloned envelope genes were prepared from
each patient plasma sample in 293 HEK cells. Viruses from individual clones
were screened for infectivity and chemokine receptor tropism in U87 cells trans-
fected with CD4 and the CCR5 or CXCR4 chemokine receptors as described
previously (69). Ten to 12 envelopes with high infectivity were selected from each
individual and evaluated in virus neutralization assays (described below).
In vitro mutagenesis. Mutations were introduced into HIV-1 envelope proteins
by site-directed mutagenesis using a QuikChange Lightning kit (Agilent, Santa
Clara, CA) followed by confirmatory sequencing. Chimeric envelope genes were
created by transferring PCR-amplified fragments between neutralization-sensi-
tive and -resistant mutants. To facilitate this transfer, novel restriction sites
preserving the virus sequence were introduced.
Antibodies and antiviral drugs. Four sera (Z23, Z1679, Z1684, and N16) from
HIV-1 infected individuals (HIV-1-positive sera) known from previous studies
(18, 59) to possess bNAbs were provided by Monogram Biosciences, Inc. (South
San Francisco, CA). Six monoclonal antibodies (MAbs) with broadly neutralizing
activity were obtained from the NIH AIDS Reagent Repository and/or Polymun
AG (Vienna, Austria). These included 2G12, b12, 17b, 2F5, 4E10, and 447D-52
(4, 9, 15, 26, 46, 64, 66, 67, 77). MAbs to the ?4?7 integrins were obtained from
two sources. The Act-1 MAb (38) was obtained from the NIH AIDS Reagent
Repository, and the ?4/VLA-4/CD49d MAb was purchased from R&D Bio-
systems (Minneapolis, MN). A cyclized synthetic peptide (CWLDVC) reported
to be a ligand for ?4?7 (2) was obtained from GenScript (Piscataway, NJ). The
antiviral compound CD4-IgG was described previously (3, 11) and provided by
GSID (South San Francisco, CA). The peptide-based antiviral drug enfuvirtide
(Fuzeon) was commercially available and produced by Roche, Inc. (Basel, Swit-
Virus neutralization assay. The study utilized a high-throughput virus neu-
tralization assay to measure the ability of monoclonal antibodies and antibodies
in HIV-1-positive plasma to inhibit infection of pseudotype viruses (17, 49, 55,
59). Briefly, pseudotype viruses were prepared by cotransfecting 293 cells with an
envelope expression vector and an envelope-deficient HIV-1 genomic vector
carrying a luciferase reporter gene. The virus-antibody mixture was incubated for
1 h prior to inoculation of U87 cells expressing CD4, CCR5, and CXCR4. Cells
were then incubated for 3 days, and then viral infectivity was measured by
luciferase expression. Neutralization data were reported as the 50% inhibitory
concentration (IC50) calculated from serum dilution curves. The positive controls
included pseudoviruses prepared from the neutralization-sensitive HIV-1 isolate
NL43 and the less neutralization-sensitive primary isolate JRCSF. The negative
virus control consisted of pseudotype viruses prepared from the envelope of the
amphotropic murine leukemia virus (aMLV). HIV-1 neutralization titers were
considered significant only if they were greater than three times the aMLV titers.
Sequence analysis. The Los Alamos HIV database (http://hiv.lanl.gov/), the
GSID HIV Data Browser (http://www.gsid.org/gsid_hiv_data_browser.html), and
the NCBI nucleotide database (http://www.ncbi.nlm.nih.gov/nuccore) were inter-
rogated to determine the degree of amino acid conservation at position 179 (180
according to HXB2 numbering). Alignments were performed using the
EMBOSS suite (54). Because of insertions and deletions, it was not practical to
identify each amino acid by use of the standard HXB2 numbering. Amino acid
positions are provided with reference to the sequences from the envelope
genes from clones 108051-005 and 108051-006 (GenBank accession numbers
HM769943 and HM769944, respectively). Wherever possible, corresponding
HXB2 numbering is provided in the text along with the 108051 numbering.
An amino acid sequence alignment of the envelope proteins from clones 005
and 006 of the 108051 virus as well as the HXB2 envelope reference sequence
is provided in Fig. S1 in the supplemental material.
In previous studies (47), we described the analysis of clade B
envelope genes obtained from a cohort of 28 individuals in-
fected with HIV-1 during the course of the VAX004 HIV
vaccine trial that ran from 1998 to 2003 (20). In these studies,
we identified seven cases where neutralization-sensitive and
neutralization-resistant clones were both observed in the same
individual. The first pair of envelopes analyzed was obtained
from subject 108060 and allowed us to identify a mutation in a
previously unexplored hydrogen-bonded ring structure that
conferred sensitivity and resistance to bNAbs. In this paper, we
report the analysis of viruses obtained from another individual
(108051) in this cohort. The envelope genes from subject
108051 were amplified by RT-PCR from cryopreserved plasma
collected at the first postdiagnosis blood draw. Envelope genes
were analyzed for infectivity and chemokine receptor usage,
and 10 envelopes with robust infectivity were isolated and
evaluated in virus neutralization assays against a panel of HIV-
1-positive sera, Z23, Z1679, Z1684, and N16, known to possess
bNAbs (59). As can be seen in Table 1, most of the clones from
patient 108051 were highly resistant to neutralization by all
four sera, with only 2 of 10 (clones 006 and 015) being sensitive
to neutralization. Based on the magnitude of the difference in
neutralization titers, we selected clones 005 and 006 for further
studies. Clone 015 gave a somewhat different pattern of neu-
tralization sensitivity and was set aside for future studies.
When we sequenced and aligned the translated gene products,
we found a total of 25 individual amino acid differences be-
tween the sensitive and resistant clones (Fig. 1A). Some dif-
ferences were due to isolated amino acid substitutions, and
TABLE 1. Neutralization of pseudoviruses containing HIV-1
envelope genes from subject 108051
Neutralizing antibody titer (IC50) for indicated human
HIV-positive serum sampleb
Z23N16 Z1684 Z1679
a“Clone” indicates the pseudotype virus prepared using the specified gp160
envelope genes. All clones tested were CCR5 tropic.
bHIV-positive sera Z23, N16, Z1684, and Z1679, known to possess broadly
neutralizing antibodies (bNAbs). The neutralizing antibody titer (IC50) is defined
as the reciprocal of the plasma dilution that produces a 50% inhibition in target
cell infection. Values in bold represent significant neutralization titers that are at
least 3 times greater than those observed for the negative-control virus (aMLV).
VOL. 84, 2010 HIV-1 NEUTRALIZATION AND THE V2 REGION11201
others represented clusters of differences resulting from dele-
tions and insertions. Further examination revealed that 16 of
the 25 amino acid differences were located in the V1 and V2
domains (Fig. 1B). To localize the amino acids responsible for
the difference in neutralization sensitivity, we systematically
transferred sequences individually and in clusters from the
sensitive clone 006 envelope protein into the resistant clone
Identification of a mutation in gp160 from subject 108051
that confers sensitivity to neutralization by HIV-1-positive sera.
When the panel of mutants was examined (Table 2), we found
that single-amino-acid substitutions at positions 272, 462, and
644 had no effect on neutralization sensitivity or resistance.
Similarly, a cluster of mutations in the cytoplasmic tail at po-
sitions 746, 748, 846, and 847 had no effect on sensitivity or
resistance. Likewise, a cluster of amino acid substitutions at the
C-terminal portion of gp120, including substitutions at posi-
tions 412, 413, and 462, had no effect on sensitivity. However,
we found that the chimeric envelope protein containing the
V1/V2 domain from clone 006 inserted into the backbone of
the 005 envelope gene markedly increased sensitivity and ex-
hibited neutralization titers comparable to those seen with the
neutralization-sensitive clone 006.
These studies located the sequences responsible for in-
creased neutralization sensitivity to either the V1 domain or
the V2 domain. We then carried out further experiments to
determine which domain was responsible (Table 2). We found
that replacement of the clone 005 V1 domain with the V1
domain from clone 006 (V1_006) did not confer increased
neutralization sensitivity. However, replacement of the V2 do-
main of clone 005 with that from clone 006 resulted in in-
creased sensitivity, similar to that of the neutralization-sensi-
tive clone 006 (Table 2). In the converse experiment, we
transferred the V1 and V2 sequences of the resistant clone 005
envelope protein into the sensitive clone 006 envelope protein.
Transferring the V1 domain preserved the neutralization-sen-
sitive phenotype, whereas transferring the V2 domain resulted
in loss of the neutralization-sensitive phenotype. Together,
these studies clearly indicated that the difference in neutral-
ization sensitivity between clones 005 and 006 could be attrib-
uted to the differences in the V2 domain.
Further studies were carried out to determine whether the
FIG. 1. Location of amino acid differences between neutralization-sensitive and -resistant clones isolated from subject 108051. HIV-1 envelope
genes were isolated from the swarm of variants in plasma from subject 108051 and tested for sensitivity and resistance to neutralization. (A) The
sequences of the neutralization-resistant clone 005 and the neutralization-sensitive clone 006 were aligned, and amino acid differences (vertical
lines) were located on the linear sequence. Conserved (C) and variable (V) domains (blue) of gp120 are indicated, as well as the locations of the
signal sequence (signal), membrane-proximal external domain (MPER), transmembrane domain (TMD), and cytoplasmic tail (CT). (B) Amino
acid sequence differences in the V1 and V2 domains between the neutralization-resistant clone 005 (contiguous sequence) and neutralization-
sensitive clone 006 (circles with arrows). Red circles indicate the locations of the D179N mutation. Open circles indicate the positions of other
amino acid substitutions. Asterisks indicate the positions of N-linked glycosylation sites.
11202 O’ROURKE ET AL. J. VIROL.
increase in neutralization sensitivity could be localized to spe-
cific amino acid substitutions in the V2 domain. As described
above, there were 11 amino acid differences in the V2 domain
between the sensitive and resistant clones of the 108051 virus.
Further mutagenesis enabled us to rule out an eight-amino-
acid insertion between positions 189 and 190 as well as single-
amino-acid changes at positions 182 and 191 (Table 2). How-
ever, the single-amino-acid substitution of asparagine (N) for
aspartic acid (D) at position 179 (corresponding to HXB2
position 180) markedly increased neutralization sensitivity and
clearly accounted for the difference in neutralization between
the neutralization-resistant clone 005 and the neutralization-
sensitive clone 006.
In these studies, it can be seen that the largest increases in
neutralization sensitivity occurred with the Z1679 and Z1684
sera, where neutralization sensitivity increased by 150-fold and
50-fold, respectively, compared to the level for the neutraliza-
tion-resistant clone 005. The effect on sensitivity to N16 and
Z23 was more moderate, with 13- and 18-fold increases, re-
spectively, possibly indicating some differences in the magni-
tude and/or specificity of particular neutralizing antibody pop-
ulations in each of the sera. The reverse mutation of N to D at
position 179 conferred neutralization resistance to the neutral-
ization-sensitive 006 clone, unambiguously confirming the im-
portance of D at position 179 in conferring the sensitive phe-
notype. The fact that multiple single-amino-acid substitutions
or clusters of substitutions at other locations within gp160 had
no effect on the neutralization phenotype showed that in-
creased neutralization sensitivity is not a trivial artifact.
Rather, only specific amino acid substitutions at specific sites
are able to convert a neutralization-resistant virus to a neu-
tralization-sensitive virus (Tables 2 and 3).
Sensitivity to neutralization by MAbs and virus entry inhib-
itors. In order to investigate the mechanism by which the
TABLE 2. Neutralization of pseudotype viruses with wild-type and
mutated envelope genes from subject 108051 by HIV-positive
sera possessing broadly neutralizing antibodies
Neutralization antibody titer (IC50)
for indicated serum sampleb
T746I, K748E, V846R,
189 insert NNNSNNN,
aWild-type resistant (wtR) and wild-type sensitive (wtS) clones from subject
108051 are indicated. The V1_006 and V2_006 designations indicate chimeric
envelope proteins in which the V1 and/or V2 domain of clone 006 replaces that
of clone 005. V1_005 and V2_005 designations indicate chimeric envelope pro-
teins in which the V1 and/or V2 domain of clone 005 replaces that of clone 006.
“?” indicates deletion.
bThe neutralizing antibody titer (IC50) is defined as the reciprocal of the
plasma dilution that produces a 50% inhibition in target cell infection. Values
in bold represent neutralization titers that are at least 3 times greater than
those observed for the negative control (aMLV). All clones tested were
TABLE 3. Sensitivity of 10851 mutants to neutralizing monoclonal antibodies and entry inhibitors
IC50(?g/ml) of indicated MAb or fusion inhibitorb
CD4-IgG 2F54E10 447-D5217b Enfuvirtide
189 insert NNNSNNN,
awtR, wild-type resistant clone; wtS, wild-type sensitive clone; ND, not done. The V1_006 and V2_006 designations indicate chimeric envelope proteins in which the
V1 and/or V2 domain of clone 006 replaces that of clone 005. The V1_005 and V2_005 designations indicate chimeric envelope proteins in which the V1 and/or V2
domain of clone 005 replaces that of clone 006. All clones tested were CCR5 tropic. The IC50s for b12 and 2G12 for all clones and mutations tested were ?20 ?g/ml.
bThe neutralizing antibody titer (IC50) is defined as the concentration of monoclonal antibodies or antiviral entry inhibitor that produces a 50% inhibition in target
cell infection. IC50values in bold print are at least 3 times greater than the IC50values measured for the specificity control virus (aMLV) and are therefore considered
positive for neutralization in this assay.
VOL. 84, 2010HIV-1 NEUTRALIZATION AND THE V2 REGION 11203
replacement of D with N at position 179 in the V2 loop alters
neutralization sensitivity, we investigated the effects of mono-
clonal antibodies (MAbs) and virus entry inhibitors that target
defined regions of the envelope protein (Table 3). These in-
cluded the b12, 17b, 2G12, and 447D-52 MAbs, known to
neutralize HIV-1 and bind to epitopes in gp120 (4, 9, 15, 26, 66,
67), and the 2F5 and 4E10 MAbs, known to bind to epitopes in
gp41 (46, 64, 77). In addition to these MAbs, we also made use
of the antiviral entry inhibitors CD4-IgG and enfuvirtide to
further define the mechanism of neutralization sensitivity. The
antiviral entry inhibitor CD4-IgG binds to the CD4 binding site
in gp120 and is able to neutralize laboratory-adapted CXCR4-
dependent isolates at low concentration (0.01 to 0.1 ?g/ml) and
primary CCR5-dependent primary isolates of HIV-1 (16) at a
high concentration (10 to 100 ?g/ml). Enfuvirtide is a peptide-
based virus entry inhibitor (39, 70) that is thought to interfere
with the formation of the six-helix bundle that is required for
virus fusion. It is thought to bind to the prehairpin intermedi-
ate structure of gp41 that is transiently formed in gp41 (45)
after binding of CD4 to the gp160 trimer (12, 23, 36).
The results obtained with this panel of inhibitors are shown
in Table 3. We found that the wild-type neutralization-resistant
clone 005 was resistant to all of the MAbs in the panel as well
as to CD4-IgG. Because of its resistance to four HIV-1-posi-
tive sera possessing bNAbs, as well as its resistance to the
broadly neutralizing MAbs 2F5, 4E10, 2G12, b12, and 447-D
and to CD4-IgG, clone 005 appears to be a tier 3 (44) neutral-
ization-resistant virus (D. Montefiori, personal communica-
tion). When we examined the properties of clone 006, we
found that it was also completely resistant to neutralization by
the b12, 2G12, and 447D-52 antibodies. Examination of the
amino acid sequences of clones 005 and 006 showed that both
envelopes contained polymorphisms in the epitopes recog-
nized by these three MAbs. The 108051 envelope proteins all
possess a GPGG sequence at the tip of the V3 loop rather than
the clade B consensus GPGR motif required for 447D-52 bind-
ing (15, 26, 63). Similarly, the 108051 residues T286, T375, and
M376 are known to be common in b12-resistant viruses and
differed from the A281, V372, and T373 (HXB2 numbering)
residues, common in b12-sensitive viruses (71, 75). Finally, the
lack of inhibition by the 2G12 antibody could be attributed to
the fact that the 108051 envelope protein lacks two of four
glycosylation sites at positions 300 and 395 (corresponding to
HXB2 positions 295 and 392) essential for binding by this
antibody (58). Thus, the resistance of clones 005 and 006 to the
b12, 2G12, and 447D-52 antibodies could be attributed to
polymorphisms at neutralizing epitopes. However, since anti-
bodies with specificities similar to those of b12 and 2G12 are
rare in HIV-1-positive sera (8, 56), another explanation was
required to account for the neutralization resistance of clone
005 to polyclonal sera (Table 1) and the remaining monoclonal
antibodies in this panel (Table 3).
First, we examined the sensitivity of the neutralization-sen-
sitive and -resistant variants to CD4-IgG (Table 3). Previous
studies (36, 37, 41) have shown that the CD4 binding site is
located entirely within the gp120 portion of the HIV-1 enve-
lope protein, recessed deeply below the apex in the native
trimer. We found that a high CD4-IgG concentration (?20
?g/ml) was required for neutralization of 108051 clone 005,
which was consistent with the concentration required to
neutralize other primary isolates of CCR5-dependent vi-
ruses (16). Replacement of D with N at position 179 in clone
005 increased sensitivity to CD4-IgG approximately 200
times compared to the level for the wild-type neutralization-
resistant clone 005 envelope. Conversely, we found that the
neutralization-sensitive clone 006 could be converted to the
CD4-IgG-resistant phenotype by replacement of N with D at
We next examined the effect of the D179N mutation on 17b,
a neutralizing MAb known to target a conserved CD4-induced
(CD4i) epitope on gp120 overlapping the coreceptor binding
region (36, 66, 67). Clone 005 was resistant to neutralization by
17b at 20 ?g/ml, and the neutralization-sensitive clone 006 was
marginally more sensitive, with an IC50of 13.3 ?g/ml (Table 3).
However, the clone 005 envelope with the D179N mutation
was approximately 5-fold more sensitive to neutralization by
this antibody. This result suggests that the D179N mutation
enhances neutralization by the 17b MAb but suggests that
other sequence differences between clone 005 and clone 006
also affect the binding of this antibody.
We next considered the effect of the D179N mutation on
sensitivity to MAbs and entry inhibitors that target sites in the
gp41 protein (Table 3). Interestingly, replacement of D with N
at position 179 in the V2 domain had a significant effect on
sensitivity to neutralization by the two broadly neutralizing
MAbs 4E10 and 2F5, directed to the membrane-proximal ex-
ternal region (MPER) of gp41. The epitopes recognized by
these antibodies are well defined, with the 2F5 MAb recogniz-
ing the ELDWA sequence and the 4E10 MAb recognizing the
adjacent NWF(D/N)IT sequence (46, 76, 77). Recent studies
suggest that the peptide in which these sequences occur is
partially embedded in the lipid bilayer (60). We found that the
D179N mutation increased neutralization sensitivity approxi-
mately 20-fold in the case of 2F5 and between 100- and 200-
fold in the case of 4E10. This result showed that a single-
amino-acid substitution in gp120 could have a dramatic effect
on the neutralizing activity of antibodies directed to the gp41
domain. Similar results were obtained with the antiviral entry
inhibitor enfuvirtide. This drug consists of a peptide derived
from gp41 sequences that overlap the MPER domain and the
C34 helix (70). The binding of enfuvirtide to gp41 is thought to
depend on CD4 binding which induces a conformational
change that exposes a binding site involving the HR1 domain
of gp41 (25). The observation that sensitivity to enfuvirtide was
increased 13-fold (Table 3) in the D179N mutant provides
additional evidence that a mutation in the V2 domain of gp120
can modulate the potency of antiviral compounds targeting the
gp41-mediated virus fusion mechanism.
Conservation of aspartic acid at position 179. Comparative
sequence analysis showed that position 179 (corresponding to
HXB2 position 180) is highly conserved across all clades of
HIV-1. We analyzed 5,918 sequences from 2,414 individuals in
three datasets, including 1,963 curated and aligned sequences
from the Los Alamos HIV Sequence database (HIV-1/SIVcpz;
2008) that listed one sequence per individual, a set of 2,908
sequences from 102 individuals with acute infections (1, 34),
and 1,047 sequences from 349 individuals with recent infec-
tions from the VAX004 HIV vaccine trial (GSID HIV Data
We found only a single naturally occurring HIV-1 sequence,
11204O’ROURKE ET AL. J. VIROL.
other than the 108051 sequence from the GSID HIV Sequence
database, where N replaced D at position 179 (GenBank ac-
cession number AF321080). Interestingly, we also found that
the D in the LDI/LDV motif was conserved in simian immu-
nodeficiency virus (SIV) and HIV-2, where it corresponded to
position 201 in the SIV reference sequence (GenBank acces-
sion number M33262). We found that this residue was con-
served in all 69 different HIV-2 and SIV sequences in the
HIV-2/SIV/MN 2008 Los Alamos HIV Sequence database.
Given the high degree of sequence variation among these
primate lentiviruses, D179 would be preserved over time and
across species only if it played an important role in the survival
of these viruses.
Further studies were carried out to try to understand the
mechanism by which aspartic acid at position 179 modulates
neutralization sensitivity in 108051. In these studies, we con-
structed a series of mutants where D at position 179 was
replaced by other amino acids (Tables 3 and 4). We found that
replacement of D at 179 with the hydrophilic, basic amino
acids arginine (R) and lysine (K) or the hydrophobic branched-
chain isoleucine (I) residue failed to yield infectious virus. This
result suggests that D179 must interact with other parts of the
envelope protein and that these interactions can alter virus
infectivity. In contrast, it was possible to replace D179 with
other amino acids that preserved virus infectivity. For example,
replacement of D with amino acids with short side chains, such
as alanine (A), serine (S), and glycine (G), resulted in infec-
tious viruses. Replacement of D179 with bulky side chains,
such as histidine (H), glutamine (Q), or the negatively charged
glutamic acid (E), also resulted in infectious viruses. However,
all of these replacements increased sensitivity to neutralization
by the polyclonal HIV-positive sera (Table 4) and the 2F5,
4E10, and 17b MAbs as well as CD4-IgG and enfuvirtide
(Table 3). Replacement of D with glutamic acid (E), whose
acidic side chain is only 1 carbon longer than D, preserved
CCR5 tropism but similarly increased neutralization sensitiv-
ity. This result indicates that there must be an extremely re-
strictive structural constraint required to preserve neutraliza-
tion resistance. Thus, the only amino acid that we have found
that can maintain the neutralization-resistant phenotype is D
at position 179.
In theory, the high level of conservation of D179 might be
critical for maintaining the conformation of the envelope pro-
tein or might be involved with receptor binding. Indeed, D179
has recently been highlighted as part of the LDV/I recognition
motif that forms the newly described ?4?7 receptor binding
site on gp120 (2). Based on this observation, we examined the
effect of ?4?7 binding inhibitors on virus neutralization in
order to determine if disruption of ?4?7 binding could account
for the observed increase in neutralization sensitivity associ-
ated with the D179N mutation. We found (see Table S1 in the
supplemental material) that neither the Act-1 MAb to ?4?7
nor the cyclic peptide inhibitor CWLDVC (2) was able to
inhibit the infectivity of JRCSF, NL43, or the wild-type neu-
tralization-sensitive and -resistant clones of 108051 in the U87
cell pseudotype neutralization assay. However, both inhibitors
(Act-1 and cyclic CWLDVC) were able to prevent the binding
of recombinant gp120 to a cell line (65) expressing ?4?7 in a
flow cytometry assay (D. Fonseca and P. Berman, unpublished
results). These results suggest that the U87 target cells used in
our assay lack the ?4?7 receptor and demonstrate that the
increased neutralization sensitivity of the D179N mutant can-
not be attributed to disruption of interactions mediated by
?4?7 in our assay system. However, this mutation might be
expected to interfere with infectivity in systems where the ?4?7
receptor is expressed on target cells.
Transfer of the D179N mutation to other viruses. To inves-
tigate possible strain-specific differences of the D179N muta-
tion on increased neutralization sensitivity, we attempted to
transfer this mutation to five other, unrelated viruses. For this
purpose, we selected four commonly used tier 2 viruses from
standard neutralization panels exhibiting a range of neutral-
ization sensitivities, specifically JRCSF, YU2, QH0692, and
TRO-11. In addition, we also examined neutralization sensi-
tivity in another virus, 108048, from the VAX004 trial. All five
viruses possessed D at the position corresponding to position
179 of the 108051 virus. Whereas all five wild-type viruses were
infectious in the U87 pseudotype assay, we found that replace-
ment of D with N at positions corresponding to 108051 posi-
tion 179 resulted in viruses with little or no infectivity. This
result suggested that D179N is essential for infectivity and
suggests that compensatory mutations may be necessary to
preserve infectivity when D is replaced by N at this position. To
further explore this possibility, we replaced the entire V1 and
V2 domains of the neutralization-resistant 108048 virus with
that of the 108051 virus containing the D179N mutation. As
can be seen in Table 4, replacement of the entire V1 and V2
domains from 108051 markedly increased sensitivity to neu-
tralization by the 4 HIV-1-positive sera, suggesting that the
compensatory mutations required to increase neutralization
sensitivity while preserving infectivity are located within the V1
or V2 domains.
TABLE 4. Envelope protein mutagenesis for investigation of the
significance of aspartic acid at position 179 in clones of the
108051 and 108048 envelope proteins
Neutralization antibody titer (IC50) for
indicated serum sampleb
Z23 N16Z1684 Z1679
aNeutralization-resistant and -sensitive clones were obtained from subject
108051 (clones 005 and 006) or from subject 108048. V1/V2_006 represents a
chimeric envelope where the V1 and V2 domains of 108051_006 replaced the V1
and V2 domains of the 108048_002 envelope. “NI” indicates no infectivity.
bThe neutralizing antibody titer (IC50) is defined as the reciprocal of the
plasma dilution that produces a 50% inhibition in target cell infection. Values in
bold represent neutralization titers that are at least 3 times greater than those
observed for the negative control (aMLV). All clones tested were CCR5 tropic.
VOL. 84, 2010HIV-1 NEUTRALIZATION AND THE V2 REGION 11205
The results presented in this study show that a single-amino-
acid mutation, D179N, in the V2 domain of gp120 can convert
a highly neutralization-resistant virus to a neutralization-sen-
sitive virus. The fact that the D179N mutation increased sen-
sitivity to neutralization by MAbs and antiviral drugs, targeting
both gp120 and gp41, suggests that the D179N mutation in-
duces a conformation change that affects accessibility of mul-
tiple neutralizing epitopes, rather than affecting the contact
residues of a single neutralizing antibody binding site. These
results suggest a far greater level of interaction between these
two subunits, with respect to neutralization sensitivity, than
was previously appreciated. The fact that D179 is conserved in
HIV-1, SIV, and HIV-2 suggests that D at position 179 may
have been preserved throughout evolution in order to preserve
resistance to neutralization by antibodies targeting epitopes in
both gp120 and gp41.
Our results are consistent with previous studies that have
identified the V2 domain of gp120 as the “global regulator of
neutralization sensitivity” (51, 52). Because the V2 domain can
be deleted entirely in some viruses while preserving virus via-
bility (10, 57, 62), it seems unlikely that the V2 domain pro-
vides a contact surface required for infectivity or virus fusion.
Rather, it appears to provide an epitope-“masking” function
that is thought to conceal important neutralizing epitopes from
neutralizing antibodies until the envelope protein undergoes a
conformational change triggered by CD4 binding (35, 36, 41).
This hypothesis is supported by studies showing increased
binding of antibodies to neutralizing epitopes in the V3 and C4
domains by envelope proteins lacking the V2 domain (10, 50,
52, 62, 72). In this regard, the single-amino-acid substitution of
N for D at position 179 appears to confer the same phenotype
as that observed when the entire V2 domain is deleted from
the SF162 virus (5, 10, 27, 61, 62, 72–74). Further data sup-
porting the role of the V2 domain in regulating neutralization
sensitivity is provided by studies showing that sensitivity and
resistance to neutralization can be transferred by moving the
V2 domain from a neutralization-sensitive virus (e.g., SF162)
onto a neutralization-resistant virus (e.g., JR-FL) backbone.
Conversely, the neutralization-sensitive SF162 virus can be
converted to a neutralization-resistant virus by exchange of the
V2 domain with that of JR-FL (52).
Although conformational masking by the V2 domain ap-
pears to explain most of the data relating to the ability of the
V2 domain to modulate neutralization sensitivity and resis-
tance (10, 43, 50, 52, 62, 72, 74), the molecular interactions
determining how the mask is “raised and lowered” have not
been characterized. Our results suggest that D179 mediates a
key interaction required for maintenance of the neutralization-
resistant, “masked” state. Replacement of D with N at position
179 seems to open up the structure of the gp160 trimer and
makes the virus more sensitive to neutralization by exposing
epitopes in both gp120 and gp41. Aspartic acid at position 179
appears to be unique, since it appears in all but two of more
than 5,918 virus sequences in the 3 datasets examined and
since all of the other mutations created in vitro at this position
resulted in either noninfectious viruses or viruses with in-
creased neutralization sensitivity. The lack of representation of
viruses with mutations at position 179 in other data sets might
reflect the fact that all other variants are noninfectious or are
so sensitive to neutralization that they are rapidly eliminated
from circulation once envelope-specific antibody responses
have developed. The fact that transfer of the D179N mutation
to five unrelated viruses (YU2, JRCSF, QH0692.42, TRO-11,
and 108048) all resulted in noninfectious viruses is consistent
with the importance of D179 in preserving the functional struc-
ture of the envelope protein and suggests that compensatory
mutations are required in other parts of the molecule to pre-
serve infectivity when D179 is replaced with N. In this regard,
the need for compensatory mutations may be similar to that
observed with V2 domain deletions where deletion of the V2
domain in the SF162 strain results in infectious viruses, whereas
deletion of the V2 domain in other strains (e.g., HXB2) requires
compensatory mutations to maintain virus infectivity (57). This
possibility is supported by the V1/V2 domain replacement ex-
periment (Table 4), where it was found that replacement of the
entire V1/V2 domain could increase sensitivity to neutraliza-
tion by HIV-1-positive sera, while preserving infectivity. With
respect to mutations at position 179, the amino acid substitu-
tions that destroyed infectivity may have stabilized the masking
function to such an extent as to prevent the conformational
changes required for infectivity following receptor binding.
Our data are also consistent with the hypothesis that the V2
masking function is dependent on quaternary interactions be-
tween the gp160 subunits that associate to form the trimeric
envelope structure that mediates virus infectivity and fusion
(13, 35, 36). Based on structural studies involving cryoelectron
tomography and X-ray data fitting, the V1 and V2 domains
appear to be located at the apex of an intermolecular contact
region within the envelope glycoprotein trimer (41). According
to this model, the native trimer is held together by strong
contacts at the gp41 base and the V1/V2 regions, with little or
no contact elsewhere. Upon CD4 binding, the monomers ro-
tate with respect to the core of the trimer to “open” the center
of the trimer, exposing CCR5 binding sites, shifting gp41 up
toward the cell membrane to form the six-helix bundle, and
exposing the fusion peptide at the target cell membrane (see
Fig. S2 in the supplemental material). When viewed in the
context of these observations, our data are consistent with the
possibility that D179 provides interactions required to main-
tain the unligated trimeric structure. Accordingly, mutations at
position 179 may weaken the quaternary, intersubunit interac-
tions, thereby providing increased access of antibodies to parts
of the molecule, such as the V3 domain, the CD4 binding site,
and the MPER, that are normally located in the interior of the
molecule and exposed only after CD4 binding. Further inves-
tigations using conformation-dependent antibodies to the V2
domain, such as the newly described PG9 and PG16 antibodies
(68), might provide additional support for this model; studies
using these antibodies as well as cryoelectron tomography are
planned to further investigate this mutation.
The results reported herein confirm and extend our previous
studies, in which swarm analysis has proved useful in identify-
ing single-amino-acid substitutions that appear to trigger con-
formational changes that expose or conceal epitopes recog-
nized by bNAbs. Envelopes with exposed neutralizing epitopes
may represent a source of immunogens potentially more ef-
fective in eliciting bNAbs than those previously tested. Enve-
lope proteins with deleted V2 domains have been tested as
11206 O’ROURKE ET AL.J. VIROL.
candidate HIV-1 vaccine antigens and were shown to elicit
higher titers of neutralizing antibodies than wild-type proteins
(5, 42, 61, 73). Studies are in progress to determine whether
immunization with the D179N mutant described in these stud-
ies exhibits broader neutralizing activity, as seen with the V2-
deleted envelope antigens.
This work was supported by a grant from the Bill & Melinda Gates
Foundation to Global Solutions for Infectious Diseases (South San
Francisco, CA) and by funding provided by the University of Califor-
nia, Santa Cruz.
We thank Julie Goss (Monogram Biosciences) for her role in project
management and Ann Durbin (UCSC) for expert technical assistance
in the preparation of the manuscript. We thank D. Burton, J. Robin-
son, S. Zolla-Pazner, H. Kattinger, and A. A. Ansari for providing
monoclonal antibodies through the NIH AIDS Research and Refer-
ence Reagent Program. We also thank I. S. Chen and Y. Yoyanagi for
the pYKJRCSF clone, B. Hahn and G. Shaw for the pYU2 clone, and
M. Li, F. Gao, and D. Montefiori for the QH0692.42 and TRO-11
envelope genes, also provided by the NIH AIDS Research and Ref-
erence Reagent Program.
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